Methods of preparing anti-human papillomavirus antigen T cells

ABSTRACT

Disclosed are methods of preparing an isolated population of human papillomavirus (HPV)-specific T cells comprise dividing an HPV-positive tumor sample into multiple fragments; separately culturing the multiple fragments; obtaining T cells from the cultured multiple fragments; testing the T cells for specific autologous HPV-positive tumor recognition; selecting the T cells that exhibit specific autologous HPV-positive tumor recognition; and expanding the number of selected T cells to produce a population of HPV-specific T cells for adoptive cell therapy. Related methods of treating or preventing cancer using the T cells are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.17/113,570, filed Dec. 7, 2020, which is a continuation of U.S.application Ser. No. 16/218,658, filed Dec. 13, 2018, now abandoned,which is a continuation of U.S. application Ser. No. 14/905,138, filedJan. 14, 2016, now abandoned, which is the U.S. national phase ofInternational Patent Application No. PCT/US2014/046478, filed Jul. 14,2014, which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/846,161, filed Jul. 15, 2013, each of which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under project numberBC010984-05 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The primary cause of some cancer types such as, for example, uterinecervical cancer, is human papillomavirus (HPV) infection. Despiteadvances in treatments such as chemotherapy, the prognosis for manycancers, including HPV-associated cancers, may be poor. Accordingly,there exists an unmet need for additional treatments for cancer,particularly HPV-associated cancers.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides a method of preparing apopulation of HPV-specific T cells, the method comprising: dividing anHPV-positive tumor sample into multiple fragments; separately culturingthe multiple fragments in the presence of only one cytokine; obtaining Tcells from the cultured multiple fragments; testing the T cells for oneor both of specific autologous HPV-positive tumor recognition and HPVantigen recognition; selecting the T cells that exhibit one or both ofspecific autologous HPV-positive tumor recognition and HPV antigenrecognition; and expanding the number of selected T cells to produce apopulation of HPV-specific T cells.

Another embodiment of the invention provides a method of preparing apopulation of HPV-specific T cells, the method comprising: dividing anHPV-positive tumor sample into multiple fragments; separately culturingthe multiple fragments; obtaining T cells from the cultured multiplefragments; testing the T cells for one or both of specific autologousHPV-positive tumor recognition and HPV antigen recognition; selectingthe T cells that exhibit one or both of specific autologous HPV-positivetumor recognition and HPV antigen recognition; and expanding the numberof selected T cells using one or both of (i) OKT3 antibody and (ii)interleukin (IL)-2 to produce a population of HPV-specific T cells.

Still another embodiment of the invention provides a method of treatingor preventing cancer in a mammal, the method comprising: dividing anHPV-positive tumor sample into multiple fragments; separately culturingthe multiple fragments; obtaining T cells from the cultured multiplefragments; testing the T cells for one or both of specific autologousHPV-positive tumor recognition and HPV antigen recognition; selectingthe T cells that exhibit one or both of specific autologous HPV-positivetumor recognition and HPV antigen recognition; expanding the number ofselected T cells to produce a population of HPV-specific T cells foradoptive cell therapy; and administering the expanded number of T cellsto the mammal in an amount effective to treat or prevent cancer in themammal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is a graph showing interferon gamma (IFN-γ) (pg/mL) secreted byeffector tumor infiltrating lymphocytes (TIL) generated from 22 tumorfragments (F1-F22) from Patient 1 or melanoma TIL upon co-culture with atarget gp100 peptide pool (shaded bars) or an HPV 18 E7 peptide pool(unshaded bars).

FIG. 1B is a graph showing IFN-γ (pg/mL) secreted by effector melanomaTIL or TIL generated from 22 tumor fragments (F1-F22) from Patient 1upon co-culture with a target autologous tumor cell line (unshaded bars)or 624 cells (a melanoma cell line) (shaded bars).

FIG. 2A is a graph showing IFN-γ (pg/mL) secreted by effector TILgenerated from tumor fragment F16, F17, or F22 of Patient 1, or by thecells given to the patient for treatment (“infusion bag”) uponco-culture with target autologous tumor cells (shaded bars), peripheralblood mononuclear cells (PBMC) from autologous tissue (forward slashhatched bars), tumor cells matched at all class I loci (backslashhatched bars), HLA-mismatched HeLa cells (vertical striped bars), orHLA-mismatched CaSki cells (horizontal striped bars).

FIG. 2B is a graph showing IFN-γ (pg/mL) secreted by effector TIL fromPatient 1 cultured alone (no tumor) or upon co-culture with autologoustumor cells that were transfected with silencing RNA against HLA-A,HLA-B, HLA-C, or RNA against an irrelevant target (non-targeting).

FIG. 2C is a graph showing IFN-γ (pg/mL) secreted by effector TIL uponco-culture with target cells (effector/targets: Patient 1 TIL/autologoustumor cells; DMFS/624 cells; or F15/HPV18E6₁₂₁₋₁₃₅) without antibody(black bars), with anti-HLA-A2 antibody (back slash hatched bars), orwith anti-Class II antibody (forward slash hatched bars). White barsindicate effector cells cultured alone.

FIG. 3 is a graph showing IFN-γ (pg/mL) secreted by effector TIL fromtumor fragment F16 or F22 of Patient 1; cells given to the patient fortreatment (“infusion bag”); melanoma TIL 1, 2, or 3 (TIL cultured frommelanoma tumors); mE7 TCR (T cells transduced to express a TCR againstHPV 16 E7₁₁₋₁₉); or F15 TIL (TIL from another patient that were reactiveagainst HPV 18 E6₁₂₁₋₁₃₅, class II-restricted, and therefore blockablewith HB145) upon co-culture with a gp100 peptide pool (white bars), OKT3antibody (horizontal striped bars), or dendritic cells (DCs) pulsed withan HPV 18 E7 peptide pool (black bars), HPV 18 E7 peptide pool and W6/32(thin forward slash hatched bars), HPV 16 E7₁₁₋₁₉ (thick forward slashhatched bars), HPV 16 E7₁₁₋₁₉ and W6/32 (checkered bars), HPV 16 E7₁₁₋₁₉and HB145 (perpendicular crossed bars), HPV 18 E6₁₂₁₋₁₃₅ (back slashhatched bars), HPV 18 E6₁₂₁₋₁₃₅ and W6/32 (crossed bars), or HPV 18E6₁₂₁₋₁₃₅ and HB 145 (bars with white-outlined black rectangles).

FIGS. 4A and 4B are graphs showing IFN-γ (pg/mL) secreted by effectorTIL clones 1-24 (A) or clones 24-48 (B) that were cloned from the F16tumor fragment from Patient 1 upon co-culture with a gp100 peptide pool(grey bars) or an HPV 18 E7 peptide pool (black bars).

FIG. 5 is a graph showing IFN-γ (pg/mL) secreted by effector TIL from 36different tumor fragments (F1-F36) from Patient 12 or melanoma TIL (DM5)(control) upon co-culture with dendritic cells pulsed with the HPV 18 E6peptide pool (black bars), the HPV 18 E7 peptide pool (grey bars) or agp100 peptide pool (control).

FIG. 6 is a graph showing IFN-γ (pg/mL) secreted by effector TILgenerated from tumor fragment F1 of Patient 12, or melanoma TIL 1, 2, or3, upon co-culture with DCs that were transduced with a lentiviralvector encoding green fluorescent protein (GFP) (white bars) or HPV 18E6 (grey bars) or pulsed with a gp100 peptide pool (forward slashhatched bars) or a HPV 18 E6 peptide pool (back slash hatched bars).Horizontal striped bars indicate TIL cultured with OKT3 antibody.

FIG. 7A is a graph showing tumor necrosis factor (TNF) a (pg/mL)secreted by clones 1, 3, 12, and 20 upon culture alone (grey bars) orwith an HPV18 E6 peptide pool (black bars).

FIG. 7B is a graph showing TNFα (pg/mL) secreted by clones 3, 12, and 20upon culture alone (white bars) or with an HPV18 E6 peptide pool (greybars) or with HPV 18 E6 peptide subpool 1 (forward slash hatched bars),subpool 2 (back slash hatched bars), subpool 3 (horizontal stripedbars), subpool 4 (vertical striped bars), or subpool 5 (black bars).

FIG. 7C is a graph showing TNFα (pg/mL) secreted by clones 3, 12, and 20upon culture alone (white bars) or with an HPV18 E6 peptide 30 (dottedbars), peptide 31 (black bars), peptide 32 (grey bars), peptide 33(vertical striped bars), peptide 34 (forward slash hatched bars),peptide 35 (back slash hatched bars), peptide 36 (squared bars), orpeptide 37 (herringbone bars).

FIGS. 8A-8C are graphs showing TNFα (pg/mL) secreted by clones 3 (A), 12(B), and 20 (C) upon co-culture with autologous PBMC (P12) or PBMC fromone of Donors 1-6.

FIG. 9A is a graph showing IFN-γ (pg/mL) secreted by clone 3 of tumorfragment F15 of Patient 12 upon co-culture with autologous PBMC (P12) orDRB1*15, DQB1*06 donor PBMC, DRB1*15 donor PBMC, or DQB1*06 donor PBMCpulsed with HPV 18 E6₇₇₋₉₁ (unshaded bars) or HPV 18 E6₁₂₁₋₁₃₅ peptide(shaded bars).

FIG. 9B is a graph showing IFN-γ (pg/mL) secreted by clone 20 of tumorfragment F15 of Patient 12 upon co-culture with PBMC pulsed with HPV 18E6₁₂₁₋₁₃₅ peptide or mF5 T cells (transduced to express anti-MART-1 TCR)co-cultured with 624 cells with antibodies against HLA-DR (horizontalstriped bars), HLA-DQ (back slash hatched bars), HLA-DP (forward slashhatched bars), pan-class I antibodies (black bars), or pan-class IIantibodies (white bars).

FIG. 10 is a graph showing IFN-γ (pg/mL) secreted by effector TIL from24 different tumor fragments (F1-F24) from Patient 4 or melanoma TIL(control) upon co-culture with dendritic cells pulsed with the HPV 16 E6peptide pool (back slash hatched bars), the HPV 16 E7 peptide pool(forward slash hatched bars) or a gp100 peptide pool (control) (blackbars). Asterisk (*) indicates TIL infused into patient.

FIG. 11A is a graph showing IFN-γ (pg/mL) secreted by CD8 positiveeffector TIL clones from Patient 4 upon co-culture with dendritic cellspulsed with an HPV 16 E7 peptide pool (shaded bars) or no peptide(unshaded bars).

FIG. 11B is a graph showing IFN-γ (pg/mL) secreted by CD4 positiveeffector TIL clones from Patient 4 upon co-culture with dendritic cellspulsed with an HPV 16 E7 peptide pool (shaded bars) or no peptide(unshaded bars).

FIG. 11C is a graph showing IFN-γ (pg/mL) secreted by CD8 positiveeffector TIL clones from Patient 4 upon co-culture with dendritic cellspulsed with an HPV 16 E6 peptide pool (shaded bars) or no peptide(unshaded bars).

FIG. 11D is a graph showing IFN-γ (pg/mL) secreted by CD4 positiveeffector TIL clones from Patient 4 upon co-culture with dendritic cellspulsed with an HPV 16 E6 peptide pool (shaded bars) or no peptide(unshaded bars).

FIGS. 12A-B are computed tomography (CT) scans of the chest of Patient 4before (A) and nine months after (B) treatment with adoptive celltherapy. The arrow in A points to a cancerous lesion in the paraaorticlymph node.

FIGS. 12C-D are CT scans of the chest of Patient 4 before (C) and ninemonths after (D) treatment with adoptive cell therapy. The arrow in Cpoints to a cancerous lesion in the left lung hilar lymph node.

FIGS. 12E-F are CT scans of the pelvis of Patient 4 before (E) and ninemonths after (F) treatment with adoptive cell therapy. The arrow in Epoints to a cancerous lesion in the common iliac lymph node.

FIG. 13 is a graph showing IFN-γ (pg/mL) secreted by effector TIL from24 different tumor fragments from Patient 8 or melanoma TIL (control)upon co-culture with dendritic cells pulsed with the HPV 18 E6 peptidepool (back slash hatched bars), the HPV 18 E7 peptide pool (forwardslash hatched bars) or a gp100 peptide pool (control) (black bars).Asterisk (*) indicates TIL infused into patient.

FIGS. 14A-B are magnetic resonance imaging (MRI) scans of the liver ofPatient 8 before (A) and two months after (B) treatment with adoptivecell therapy. The arrow in A points to a cancerous liver mass.

FIGS. 14C-D are CT scans of the abdomen of Patient 8 before (C) and twomonths after (D) treatment with adoptive cell therapy. The arrow in Cpoints to a cancerous lesion in the retroperitoneal lymph node.

FIGS. 14E-F are CT scans of the abdomen of Patient 8 before (E) and twomonths after (F) treatment with adoptive cell therapy. The arrow in Epoints to a cancerous abdominal wall mass.

FIGS. 14G-H are CT scans of the pelvis of Patient 8 before (G) and twomonths after (H) treatment with adoptive cell therapy. The arrow in Gpoints to a cancerous left pericolic mass.

FIG. 15A is a graph showing paraaortic (circles), left hilar (squares),right hilar (Δ), and common iliac (∇) tumor size measurement (% changefrom baseline) of Patient 4 at time points (number of months) afterHPV-TIL infusion.

FIG. 15B is a graph showing abdominal wall (circles), paraaortic(squares), left pelvis (Δ), and right ureter (∇) tumor size measurement(% change from baseline) of Patient 8 at time points (number of months)after HPV-TIL infusion.

FIGS. 16A-D are delayed gadolinium-enhanced T1-weighted MRI images ofPatient 8. A and C each show a tumor on the liver surface beforetreatment. B and D show that neither tumor was present 11 monthsfollowing treatment. Arrows in A and C indicate locations of the tumors.

FIG. 17A is a graph showing the serum cytokine level (pg/ml) measured inPatient 4 at time points (number of days) after treatment. Cytokinesmeasured include interleukin (IL)-2 (closed circles), IL-4 (squares),IL-6 (▴), IL-13 (▾), granulocyte colony-stimulating factor (G-CSF)(diamonds), and tumor necrosis factor alpha (TNF-a) (open circles).Cytokines that were administered to the patient are underlined.

FIG. 17B is a graph showing the serum cytokine level (pg/ml) measured inPatient 8 at number of days after treatment. Cytokines measured includeIL-2 (closed circles), IL-5 (closed squares), IL-6 (▴), IL-8 (▾), G-CSF(diamonds), monocyte chemoattractant protein-1 (MCP-1) (open circles),and TNF-a (open squares). Cytokines that were administered to thepatient are underlined.

FIGS. 18A-D are graphs showing the reactivity of TIL to be administeredto Patient 4 (A and C) or Patient 8 (B and D) (shaded bars) against HPVE6, HPV E7, or Epstein Barr virus (EBV) (control) as measured byIFN-gamma secretion (pg/ml) (A and B) and ELISPOT assays (C and D).Unshaded bars represent the reactivity of EBV-reactive T cells from thesame patient (control).

FIGS. 19A-B are graphs showing lymphocyte counts (cells/mm³) for Patient4 (A) and Patient 8 (B) at various time points (days) after infusion ofTIL. Cells counted include CD8 T cells (squares), CD4 T cells (circles),NK cells (▴), and B cells (▾).

FIGS. 19C-D are graphs showing HPV-reactive T cells detected inperipheral blood of Patient 4 (C) or Patient 8 (D) at various timepoints (months) after infusion of TIL as measured by IFN-gamma (pg/ml).Reactivity against HPV E6 (grey bars), HPV E7 (black bars), or gp100(control) (unshaded bars) was measured.

FIGS. 19E-F are graphs showing the quantification of HPV-reactive Tcells detected in peripheral blood of Patient 4 (E) or Patient 8 (F) atvarious time points (months) after infusion of TIL as measured byELISPOT. Reactivity against HPV E6 (grey bars), HPV E7 (black bars), orgp100 (control) (unshaded bars) was measured.

FIGS. 20A-J are magnetic resonance imaging (MRI) scans of Patient 13 whohad a metastatic tonsil cancer before (A, C, E, G, and I) and fourmonths after (B, D, F, H, and J) treatment with adoptive cell therapy.The arrows point to multiple malignant tumors in the lungs and the rightlung hilum.

FIGS. 21A-B are CT scans of the chest of Patient 4 before (A) and 18months after (B) treatment with adoptive cell therapy. The arrow in Apoints to a cancerous lesion in a paraaortic lymph node.

FIGS. 21C-D are CT scans of the chest of Patient 4 before (C) and 18months after (D) treatment with adoptive cell therapy. The arrows in Cpoint to a left hilar lesion and a subcarinal lesion.

FIGS. 21E-F are CT scans of the chest of Patient 4 before (E) and 18months after (F) treatment with adoptive cell therapy. The arrows in Epoint to bilateral hilar lesions.

FIGS. 21G-H are CT scans of the pelvis of Patient 4 before (G) and 18months after (H) treatment with adoptive cell therapy. The arrow in Gpoints to a cancerous lesion in the common iliac lymph node.

FIGS. 22A-B are CT scans of the abdomen of Patient 8 before (A) and 11months after (B) treatment with adoptive cell therapy. The arrow in Apoints to a cancerous lesion in a retroperitoneal lymph node.

FIGS. 22C-D are CT scans of the abdomen of Patient 8 before (C) and 11months after (D) treatment with adoptive cell therapy. The arrows in Cpoint to a cancerous abdominal wall mass and a retroperitonal tumor.

FIGS. 22E-F are CT scans of the abdomen of Patient 8 before (E) and 11months after (F) treatment with adoptive cell therapy. The arrow in Epoints to a cancerous paracolic mass.

FIGS. 22G-H are CT scans of the pelvis of Patient 8 before (G) and 11months after (H) treatment with adoptive cell therapy. The arrow in Gpoints to a cancerous left pelvic mass. The triangle in H points to anuretal stent.

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that populations of human papillomavirus(HPV)-specific T cells can be prepared for a variety of applications,for example, adoptive cell therapy. The inventive methods may generatecells that are useful for treating a variety of conditions, e.g.,cancer.

The inventive methods provide numerous advantages. For example, theinventive methods may, advantageously, generate T cells fromHPV-positive cancers at a grade and scale suitable for clinical use.Additionally, the inventive methods may, advantageously, generate Tcells that recognize the HPV antigens E6 and E7, which areconstitutively and specifically expressed by cancer cells and are notexpressed by normal cells. Therefore, without being bound to aparticular theory or mechanism, it is believed that T cells generated bythe inventive methods advantageously target the destruction of cancercells while minimizing or eliminating the destruction of normal,non-cancerous cells, thereby reducing, for example, by minimizing oreliminating, toxicity. In addition, because an embodiment of theinventive methods includes nonmyeloablative chemotherapy, the inventivemethods can advantageously be used to treat patients that would not beeligible for treatments that involve total body irradiation (TBI) suchas, for example, patients that have already undergone myeloablativetherapy, e.g., radiotherapy, prior to treatment; patients with comorbidconditions; and patients with less than 2×10⁶ CD34⁺ cells/kg. Moreover,the inventive methods of treating cancer may, advantageously,successfully treat or prevent HPV-positive cancers that do not respondto other types of treatment such as, for example, chemotherapy alone,surgery, or radiation.

An embodiment of the invention comprises obtaining an HPV-positive tumorsample from a mammal. The tumor sample may be obtained from a mammal inany suitable manner such, for example, biopsy or surgical resection.

In an embodiment, the method may comprise testing the tumor sample forHPV infection. The HPV may be any HPV subtype. Preferably, the HPVsubtype is HPV 16 or HPV 18. The testing may comprise testing for theexpression of any protein (e.g., an antigen) specifically expressed byHPV-infected cells such as, for example, any one or more of HPV 16 E6,HPV 16 E7, HPV 18 E6, and HPV 18 E7, expression of any RNA encoding theHPV-specific protein, or a combination thereof. Testing for HPVinfection may be carried out in any suitable manner known in the art.Exemplary HPV tests may include any one or more of reverse transcriptase(RT) polymerase chain reaction (PCR)-based genotyping and Western blots.The tumor sample may be positive for any subtype of HPV infection suchas, for example, HPV 16 or HPV 18 infection.

An embodiment of the invention comprises dividing the HPV-positive tumorsample into multiple fragments. The tumor sample may be divided into anysuitable number of fragments such as, for example, 4, 8, 12, 16, 20, 24,28, 32, 36, 40 or more fragments. Preferably, the tumor sample isdivided into 24 fragments. The tumor sample may be divided in anysuitable manner e.g., mechanically (disaggregating the tumor using,e.g., a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif.) orenzymatically (e.g., collagenase or DNase).

An embodiment of the invention comprises separately culturing themultiple fragments. In this regard, the fragments may be cultured inseparate containers, e.g., separate plates or separate wells of a plate.The multiple fragments may be cultured in any suitable manner. Forexample, the fragments may be cultured in a gas permeable container asdescribed in U.S. Patent Application Publication No. 2012/0244133. In anembodiment of the invention, the tumor fragments are cultured in thepresence of a combination of two or more cytokines. In a preferredembodiment, however, the method comprises culturing the tumor fragmentsin the presence of only one cytokine. The cytokine may be any suitablecytokine such as, for example, interleukin (IL)-2, IL-7, IL-15, orIL-12. Preferably, the cytokine is IL-2. The tumor fragments may becultured in any suitable amount of cytokine (e.g., from about 30 IU/mLto about 6,000 IU/mL, preferably about 6,000 IU/mL). Preferably, themethod comprises culturing tumor fragments in about 6,000 IU/mL IL-2.

The method may comprise obtaining T cells from the cultured multiplefragments. The method may comprise culturing the T cells untilconfluence (e.g., about 2×10⁶ lymphocytes per mL in a 24-well plate),e.g., from about 12 to about 28 days.

The method may comprise testing the T cells for one or both of specificautologous HPV-positive tumor recognition and HPV antigen recognition.Specific autologous HPV-positive tumor recognition can be tested by anymethod known in the art, e.g., by measuring cytokine release (e.g.,interferon (IFN)-y) following co-culture with autologous HPV-positivetumor cells. T cells may be considered to recognize HPV-positive tumorif, for example, co-culture with autologous HPV-positive tumor cellsresults in IFN-γ release that is one or more of (i) twice the amount ofIFN-γ that is measured when the T cells are cultured alone (background);(ii) at least about 200 pg/mL or more (e.g., 200 pg/mL or more, 300pg/mL or more, 400 pg/mL or more, 500 pg/mL or more, 600 pg/mL or more,700 pg/mL or more, 1000 pg/mL or more, 5,000 pg/mL or more, 7,000 pg/mLor more, 10,000 pg/mL or more, or 20,000 pg/mL or more); and (iii)blocked by MHC Class I antibody by greater than about 40%, greater thanabout 50%, or greater than about 60%.

Specific HPV antigen recognition can be tested by any method known inthe art, e.g., by measuring cytokine release (e.g., IFN-γ) followingco-culture with antigen-negative antigen presenting cells (e.g.,dendritic cells) that have been pulsed with a peptide of an HPV antigen.T cells may be considered to recognize HPV antigen if, for example,IFN-γ release is one or both of (i) twice the amount of IFN-γ that ismeasured when the T cells are cultured with antigen presenting cellsthat are pulsed with a negative control peptide and (ii) at least about200 pg/mL or more (e.g., 200 pg/mL or more, 300 pg/mL or more, 400 pg/mLor more, 500 pg/mL or more, 600 pg/mL or more, 700 pg/mL or more, 1000pg/mL or more, 5,000 pg/mL or more, 7,000 pg/mL or more, 10,000 pg/mL ormore, or 20,000 pg/mL or more) of IFN-γ upon co-culture withantigen-negative antigen presenting cells pulsed with a lowconcentration of HPV 16 or HPV 18 peptide (e.g., about 0.05 ng/mL toabout 5 ng/mL, 0.05 ng/mL, 0.1 ng/mL, 0.5 ng/mL, 1 ng/mL, or 5 ng/mL).The T cells may also secrete IFN-γ upon co-culture with antigen-negativeantigen presenting cells pulsed with higher concentrations of HPVpeptide.

The HPV antigen may be any HPV antigen. For example, the HPV antigen maybe any one or more of HPV 16 E6, HPV 16 E7, HPV 18 E6, and HPV 18 E7.While in some embodiments, the population of T cells may specificallyrecognize only one HPV antigen, in some embodiments, the population of Tcells may specifically recognize more than one HPV antigen. In thisregard, the population of T cells may comprise multiple T cells eachhaving different HPV specificities. For example, the population of Tcells may include some T cells that specifically recognize HPV 16 E6 andother T cells that specifically recognize HPV 16 E7, or the populationmay include some T cells that specifically recognize HPV 18 E6 and otherT cells that specifically recognize HPV 18 E7.

The method may comprise selecting the T cells that exhibit one or bothof specific autologous HPV-positive tumor recognition and HPV antigenrecognition. In an embodiment of the invention, while testing the Tcells for one or both of specific autologous HPV-positive tumorrecognition and HPV antigen recognition may identify those cultures thatcontain T cells that recognize HPV, those cultures that contain theHPV-reactive T cells may also contain additional T cells that arereactive against other, non-HPV tumor antigens. Accordingly, theselected population of T cells may include polyclonal T cells withmultiple specificities. In another embodiment of the invention, thetesting identifies cultures that only contain T cells that recognizeHPV. In this regard, the selected population of T cells may include Tcells with only HPV specificity.

The method may further comprise expanding the number of selected T cellsto produce a population of HPV-specific T cells. Rapid expansionprovides an increase in the number of antigen-specific T-cells of atleast about 100-fold (or 200-, 300-, 400-, 500-, 600-, 700-, 800-,900-fold, or greater) over a period of about 10 to about 14 days,preferably about 14 days. More preferably, rapid expansion provides anincrease of at least about 1000-fold (or 1500-, 2000-, 2500-, 3000-fold,or greater) over a period of about 10 to about 14 days, preferably about14 days. Most preferably, rapid expansion provides an increase of atleast about 1000-fold to about 3000-fold over a period of about 10 toabout 14 days, preferably about 14 days.

Expansion of the numbers of T cells can be accomplished by any of anumber of methods as are known in the art as described in, for example,U.S. Pat. Nos. 8,034,334; 8,383,099; U.S. Patent Application PublicationNo. 2012/0244133; Dudley et al., J. Immunother., 26:332-42 (2003); andRiddell et al., J. Immunol. Methods, 128:189-201 (1990). For example,the numbers of T cells can be rapidly expanded using non-specific T-cellreceptor stimulation in the presence of feeder lymphocytes and eitherinterleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 beingpreferred. The non-specific T-cell receptor stimulus can include around30 ng/mL of OKT3, a mouse monoclonal anti-CD3 antibody (available fromOrtho-McNeil, Raritan, N.J.). Alternatively, the number of T cells canbe rapidly expanded by stimulation in vitro with an antigen (one ormore, including antigenic portions thereof, such as epitope(s), or acell) of the cancer, which can be optionally expressed from a vector,e.g., 0.3 μM MART-1:26-35 (27L) or gp100:209-217 (210M), in the presenceof a T-cell growth factor, such as 300 IU/mL IL-2 or IL-15, with IL-2being preferred. The numbers of in vitro-induced T-cells may be rapidlyexpanded by re-stimulation with the same antigen(s) of the cancer pulsedonto antigen-presenting cells. In an embodiment, the numbers of T cellsare expanded in a gas permeable container as described in U.S. PatentApplication Publication No. 2012/0244133.

In an embodiment of the invention, the method comprises expanding thenumber of T cells using one or both of (i) irradiated allogeneic feedercells and (ii) irradiated autologous feeder cells and one or both of(iii) OKT3 antibody and (iv) a T-cell growth factor, such as IL-2 orIL-15, with IL-2 being preferred. The method may comprise expanding thenumber of T cells using one or both of (i) irradiated allogeneic feedercells and (ii) irradiated autologous feeder cells and one or both of(iii) OKT3 antibody and (iv) interleukin (IL)-2. Preferably, the methodcomprises expanding the number of T cells using one or both of (i)irradiated allogeneic feeder cells and (ii) irradiated autologous feedercells and both of (iii) OKT3 antibody and (iv) interleukin (IL)-2. In anespecially preferred embodiment, the method comprises expanding thenumber of T cells using (i) irradiated allogeneic feeder cells, (ii)OKT3 antibody and (iii) interleukin (IL)-2.

In still another embodiment, the method comprises expanding the numberof selected T cells using one or both of (i) OKT3 antibody and (ii)interleukin (IL)-2 to produce a population of HPV-specific T cells,optionally in combination with one or both of irradiated allogeneicfeeder cells and irradiated autologous feeder cells. In this regard, anembodiment of the invention provides a method of preparing a populationof HPV-specific T cells, the method comprising: dividing an HPV-positivetumor sample into multiple fragments; separately culturing the multiplefragments; obtaining T cells from the cultured multiple fragments;testing the T cells for one or both of specific autologous HPV-positivetumor recognition and HPV antigen recognition; selecting the T cellsthat exhibit one or both of specific autologous HPV-positive tumorrecognition and HPV antigen recognition; and expanding the number ofselected T cells using one or both of (i) OKT3 antibody and (ii)interleukin (IL)-2 to produce a population of HPV-specific T cells.Dividing the tumor sample, culturing the tumor fragments, obtaining Tcells, testing the T cells, selecting the T cells, and expanding thenumbers of selected T cells may be carried out as described herein withrespect to other aspects of the invention.

The population of expanded numbers of T cells produced by the inventivemethods may specifically recognize HPV-positive cells, e.g.,HPV-positive cancer cells. The cells that are recognized by the T cellsmay be positive for any subtype of HPV such as, for example, HPV 16 orHPV 18. Alternatively or additionally, the population of T cellsproduced by the inventive methods may specifically recognize any HPVantigen such as, for example, any one or more of HPV 16 E6, HPV 16 E7,HPV 18 E6, and HPV 18 E7. While in some embodiments, the population of Tcells may specifically recognize only one HPV antigen, in someembodiments, the population of T cells may specifically recognize morethan one HPV antigen. In this regard, the population of expanded numbersof T cells may comprise multiple T cells each having different HPVspecificities. For example, the population of expanded numbers of Tcells may include some T cells that specifically recognize HPV 16 E6 andother T cells that specifically recognize HPV 16 E7, or the populationmay include some T cells that specifically recognize HPV 18 E6 and otherT cells that specifically recognize HPV 18 E7. The ability of thepopulation of expanded numbers of T cells produced by the inventivemethods to specifically recognize HPV-positive cells and to specificallyrecognize a HPV antigen may be measured as described herein with respectto other aspects of the invention.

The population of T cells produced by the inventive methods may beuseful for treating or preventing HPV-associated conditions, e.g.,cancer. Accordingly, another embodiment of the invention provides amethod of treating or preventing cancer in a mammal, the methodcomprising preparing a population of HPV-specific T cells according toany of the inventive methods described herein and administering thepopulation of T cells to the mammal in an amount effective to treat orprevent cancer in the mammal.

Another embodiment of the invention provides a method of treating orpreventing cancer in a mammal, the method comprising: dividing anHPV-positive tumor sample into multiple fragments; separately culturingthe multiple fragments; obtaining T cells from the cultured multiplefragments; testing the T cells for one or both of specific autologousHPV-positive tumor recognition and HPV antigen recognition; selectingthe T cells that exhibit one or both of specific autologous HPV-positivetumor recognition and HPV antigen recognition; expanding the number ofselected T cells to produce a population of HPV-specific T cells foradoptive cell therapy; and administering the expanded number of T cellsto the mammal in an amount effective to treat or prevent cancer in themammal. Dividing the tumor sample, culturing the tumor fragments,obtaining T cells, testing the T cells, selecting the T cells, andexpanding the numbers of selected T cells may be carried out asdescribed herein with respect to other aspects of the invention. Anotherembodiment of the invention provides a method of treating or preventinga condition in a mammal, the method comprising preparing a population ofHPV-specific T cells according to any of the inventive methods describedherein and administering the population of T cells to the mammal in anamount effective to treat or prevent the condition in the mammal whereinthe condition is cancer, HPV 16 infection, or HPV-positivepremalignancy.

An embodiment of the invention comprises administering to the mammalnonmyeloablative lymphodepleting chemotherapy. The nonmyeloablativelymphodepleting chemotherapy can be any suitable such therapy, which canbe administered by any suitable route. The nonmyeloablativelymphodepleting chemotherapy can comprise the administration ofcyclophosphamide and fludarabine, particularly if the cancer is anHPV-positive cancer, which can be metastatic. A preferred route ofadministering cyclophosphamide and fludarabine is intravenously.Likewise, any suitable dose of cyclophosphamide and fludarabine can beadministered. Preferably, around 60 mg/kg of cyclophosphamide isadministered for two days after which around 25 mg/m² fludarabine isadministered for five days, particularly if the cancer is anHPV-positive cancer. In an embodiment of the invention, thenonmyeloablative lymphodepleting chemotherapy is administered prior toadministering the T cells.

An embodiment of the invention comprises, after administering thenonmyeloablative lymphodepleting chemotherapy, administering to themammal the population of HPV-specific T cells prepared by any of theinventive methods described herein.

The T-cells can be administered by any suitable route as known in theart. Preferably, the T-cells are administered as an intra-arterial orintravenous infusion, which preferably lasts about 30 to about 60minutes. Other examples of routes of administration includeintraperitoneal, intrathecal and intralymphatic.

Likewise, any suitable dose of T-cells can be administered. Preferably,from about 1.0×10¹⁰ T-cells to about 13.7×10¹⁰ T-cells are administered,with an average of around 5.0×10¹⁰ T-cells, particularly if the canceris an HPV-positive cancer. Alternatively, from about 1.2×10¹⁰ to about4.3×10¹⁰ T-cells are administered.

In an embodiment of the invention, any of the methods described hereinmay further comprise combining the population of HPV-specific T cellswith a pharmaceutically acceptable carrier. The pharmaceuticallyacceptable carrier may be any pharmaceutically acceptable carrier thatis suitable for adoptive cell therapy. For example, the pharmaceuticallyacceptable carrier may include any isotonic carrier such as, forexample, normal saline (about 0.90% w/v of NaCl in water, about 300mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOLR electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter,Deerfield, Ill.), about 5% dextrose in water, or Ringer's lactate. In anembodiment, the pharmaceutically acceptable carrier is supplemented withhuman serum albumen.

An embodiment of the invention comprises enriching cultured T cells forCD8⁺ T cells prior to rapid expansion of the cells. Following culture ofthe T cells, the T cells are depleted of CD4⁺ cells and enriched forCD8⁺ cells using, for example, a CD8 microbead separation (e.g., using aCliniMACS^(plus) CD8 microbead system (Miltenyi Biotec)). Without beingbound to a particular theory, it is believed that CD8⁺ enrichment ofsome T cell cultures reveals in vitro tumor recognition that may not beevident in the bulk culture, and improved in vitro recognition of tumorin other cultures. Additionally, the enriched CD8⁺ T cells are believedto behave more reliably and predictably in clinical scale rapidexpansions than the bulk T cells.

An embodiment of the invention comprises enriching cultured T cells forCD4⁺ T cells prior to rapid expansion of the cells. Following culture ofthe T cells, the T cells are depleted of CD8⁺ cells and enriched forCD4⁺ cells using, for example, a CD4 microbead separation (e.g., using aCliniMACS^(plus) CD8 microbead system (Miltenyi Biotec)).

In an embodiment, a T-cell growth factor that promotes the growth andactivation of the autologous T cells is administered to the mammaleither concomitantly with the autologous T cells or subsequently to theautologous T cells. The T-cell growth factor can be any suitable growthfactor that promotes the growth and activation of the autologousT-cells. Examples of suitable T-cell growth factors include interleukin(IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in variouscombinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15,IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.IL-12 is a preferred T-cell growth factor.

In an embodiment, the autologous T-cells are modified to express aT-cell growth factor that promotes the growth and activation of theautologous T-cells. Suitable T-cell growth factors include, for example,any of those described above. Suitable methods of modification are knownin the art. See, for instance, Green and Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. 2012; and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, N Y, 1994.Desirably, modified autologous T-cells express the T-cell growth factorat high levels. T-cell growth factor coding sequences, such as that ofIL-12, are readily available in the art, as are promoters, the operablelinkage of which to a T-cell growth factor coding sequence promotehigh-level expression.

The T-cell growth factor can be administered by any suitable route. Ifmore than one T-cell growth factor is administered, they can beadministered simultaneously or sequentially, in any order, and by thesame route or different routes. Preferably, the T-cell growth factor,such as IL-2, is administered intravenously as a bolus injection. Thedosage of the T-cell growth factor may be chosen based on patienttolerance. For example, the T-cell growth factor may be administereduntil one or more limiting adverse events occur. Desirably, the dosageof the T-cell growth factor, such as IL-2, is what is considered bythose of ordinary skill in the art to be high. Preferably, a dose ofabout 720,000 IU/kg of IL-2 is administered three times daily untiltolerance, particularly when the cancer is an HPV-positive cancer.Preferably, about 5 to about 15 doses of IL-2 are administered, with anaverage of around 9 doses.

In an embodiment, the autologous T-cells may be modified to express a Tcell receptor (TCR) having antigenic specificity for an HPV antigen,e.g., any of the HPV antigens described herein. Suitable methods ofmodification are known in the art. See, for instance, Green and Sambrookand Ausubel, supra. For example, the T cells may be transduced toexpress a T cell receptor (TCR) having antigenic specificity for an HPVantigen using transduction techniques described in Heemskerk et al. HumGene Ther. 19:496-510 (2008) and Johnson et al. Blood 114:535-46 (2009).

With respect to the inventive methods, the cancer can be any cancer,including any of acute lymphocytic cancer, acute myeloid leukemia,alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer,cancer of the anus, anal canal, or anorectum, cancer of the eye, cancerof the intrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vagina, cancer of the vulva,chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer,esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor,glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynxcancer, liver cancer, lung cancer, malignant mesothelioma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer ofthe oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer, skin cancer, small intestinecancer, soft tissue cancer, stomach cancer, testicular cancer, thyroidcancer, cancer of the uterus, ureter cancer, and urinary bladder cancer.A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is HPV-positive cancer. The HPV-positivecancer may be, for example, HPV 16-positive or HPV 18-positive cancer.While the cancers most commonly associated with HPV infection includecancer of the uterine cervix, oropharynx, anus, anal canal, anorectum,vagina, vulva, and penis, the inventive methods may be used to treat anyHPV-positive cancer, including those that occur at other anatomicalareas.

As used herein, the term “mammal” refers to any mammal, including, butnot limited to, mammals of the order Rodentia, such as mice andhamsters, and mammals of the order Logomorpha, such as rabbits. It ispreferred that the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs). It is more preferred that the mammalsare from the order Artiodactyla, including Bovines (cows) and Swines(pigs) or of the order Perssodactyla, including Equines (horses). It ismost preferred that the mammals are of the order Primates, Ceboids, orSimoids (monkeys) or of the order Anthropoids (humans and apes). Anespecially preferred mammal is the human.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. For example, the treatment or prevention provided by theinventive method can include promoting the regression of a tumor. Also,for purposes herein, “prevention” can encompass delaying the onset ofthe disease, or a symptom or condition thereof.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates a method of preparing HPV-positivetumor-infiltrating lymphocytes (TIL) for adoptive cell therapy.

Patients were entered into clinical protocols and signed informedconsents that were approved by the Institutional Review Board of theNational Cancer Institute prior to tumor resection. Tumors were excisedfrom patients. Tumors were tested for HPV 16 E6, HPV 16 E7, HPV 18 E6,and HPV 18 E7 expression using reverse transcriptase (RT) polymerasechain reaction (PCR) genotyping.

Multiple (24) independent cultures of HPV 16 E6 positive, HPV E7positive, HPV 18 E6 positive, and HPV E7 positive TIL were set up usingenzymatic digests and tumor fragments (1-2 mm³) procured by sharpdissection. TIL from tumor digests were generated by culturingsingle-cell suspensions (5×10⁵/mL) obtained by overnight enzymaticdigestion of tumor fragments in media containing collagenase,hyaluronidase, and DNAse. Cultures of tumor fragments and digests wereinitiated in 2 mL wells of complete medium (CM) and IL-2 (6000 IU/mL,Chiron Corp., Emeryville, Calif.) in a humidified 37° C. incubator with5% CO2. CM included RPMI1640 with glutamine, plus 10% human AB serum, 25mM HEPES, 10 μg/mL gentamicin, and 5.5×10⁻⁵M 2-mercaptoethanol. Fivedays after initiation, one half of the media was aspirated from thewells and replaced with fresh CM and IL-2, and media was replaced everytwo to three days thereafter as needed. Under these conditions,lymphocytes will first lyse the adherent cells in the well, and thenbegin to multiply and grow.

TIL cultures achieved confluent growth of the original 2-mL well andeliminated adherent tumor cells, typically about 12-28 days afterinitiation. In practice, this was about 4×10⁶ lymphocytes from eachoriginal tumor fragment or digest well. By pooling all the wells in asingle 24 well plate, approximately 5×10⁷ TIL cells would be obtained.

When cultures designated for TIL generation expanded to confluence in2-mL wells, they were tested for HPV specific reactivity. Because theTIL were set up in large numbers (typically groups of 24 per tumor) itwas not feasible to count each TIL culture individually. The TILspecificity assay measures activity per volume rather than activity percell. Each well was mixed thoroughly, and one hundred microliters oflymphocytes (estimated 1×10⁵ cells) were washed and cocultured overnightwith autologous tumor digest or autologous monocyte-derived dendriticcells (DCs) pulsed with HPV 16 and HPV 18 E6 and E7 MACS PEPTIVATORpeptide pools. The peptide pools included 15-mer peptides with11-amino-acid overlaps that covered the complete sequence of E6 or E7(HPV 16 or HPV 18). The peptide pools were over 75% pure and low inendotoxin. IFN-γ release was then measured with enzyme-linkedimmunosorbent assay (ELISA). The results are shown in Table A.

TABLE A Peptide Tumor digest reactivity reactivity Primary (number(number Patient Site of of tumor of tumor Number Cancer HPV typefragments)¹ fragments)²  1 cervix HPV-18 E7 (22) Yes (4)  2 tonsilHPV-16 E7 (5) —  3 cervix HPV-18 E6 (7), E7 (19) Yes (3)  4 cervixHPV-16 E6 (18), E7 (23) No  5 cervix HPV-16 E6 (1) No  6 unknown (neck)HPV-16 E6 (23), E7 (22) —  7 cervix HPV-18 E6 (1), E7 (5) —  8 cervixHPV-18 E7 (3) —  9 cervix HPV-18 none No 10 unknown (pelvic) HPV-16 E6(1) Yes (1) 11 cervix HPV-18 none No 12 cervix HPV-18 E6, E7 — 13 anusHPV-16 none — 14 anus HPV-16 E6 — 15 cervix HPV-16 E6, E7 — ¹Defined asat least one tumor fragment with >200 pg/mL and twice background(negative control peptide pool). ²Defined as >200 pg/mL IFN-γ, twicebackground (TIL alone), and >50% blocking with W6/32 MHC class 1Antibody. “—”indicates not tested.

Rapid expansion of the numbers of HPV reactive TIL were performed usingthe Rapid Expansion Protocol (REP) as previously described (Dudley etal., J. Immunother., 26:332-42 (2003) and Riddell et al., J. Immunol.Methods, 128:189-201 (1990)). Briefly, TIL cells were cultured in gaspermeable, G-REX flasks with a 200 fold excess of irradiated (40 Gy)allogeneic peripheral blood mononuclear “feeder” cells in completemedium (CM) with 30 ng/mL anti-CD3 antibody and 6000 IU/mL IL-2. Half ofthe media was exchanged on day 5 using CM with 6000 IU/mL IL-2, andcells were split as needed thereafter. TIL expanded an average of morethan 3000 fold.

Example 2

This example demonstrates the reactivity of the TIL from Patient 1.

TIL were generated as described in Example 1 from 22 different tumorfragments (F1-F22) from Patient 1. The TIL from Patient 1 or melanomaTIL (control) were co-cultured with dendritic cells pulsed with the HPV18 E7 peptide pool or a gp100 peptide pool (control) and IFN-γ wasmeasured. The results are shown in FIGS. 1A-1B. As shown in FIGS. 1A-1B,the TIL from tumor fragment 22 of Patient 1 recognized an autologoustumor line but not HPV 18 E7 peptides.

The TIL from tumor fragments F16, F17, or F22 of Patient 1 or cellsgiven to the patient for treatment (“infusion bag”) were co-culturedwith autologous tumor, peripheral blood mononuclear cells (PBMC) fromautologous tissue, tumor cells matched at all class I loci, HeLa cells(HLA mismatched), or CaSki cells (HLA mismatched). IFN-γ was measured.The results are shown in FIG. 2A. As shown in FIG. 2A, TIL from tumorfragments F16 and F22 showed autologous tumor recognition.

Autologous tumor cells were transfected with silencing RNA againstHLA-A, HLA-B, HLA-C, or irrelevant RNA (non-targeting) and wereco-cultured with TIL from Patient 1. IFN-γ was measured. The results areshown in FIG. 2B. As shown in FIG. 2B, recognition of TIL from Patient 1was diminished by HLA-A silencing.

Effector/target cells (Patient 1 TIL/autologous tumor cells; DMFS/624cells; or Patient 12 (P12) F15/HPV18E6₁₂₁₋₁₃₅) were cultured alone orco-cultured without antibody, with anti-HLA-A2 antibody, or anti-ClassII antibody. DMFS cells are T cells transduced to express a MHC classI-restricted TCR against MART-1. The results are shown in FIG. 2C. Asshown in FIG. 2C, recognition of TIL from Patient 1 was not inhibited byHLA-A*02 blocking, which suggested HLA-A*01 restricted tumorrecognition. Patient 1's haplotype was HLA-A*01, HLA-A*02.

TIL from tumor fragment F16 or F22 of Patient 1; cells given to thepatient for treatment (“infusion bag”); melanoma TIL 1, 2, or 3 (TILcultured from melanoma tumors); mE7 TCR (T cells from PBMC that weretransduced to express a TCR against HPV 16 E7₁₁₋₁₉); or F15 TIL (TILfrom another patient that were reactive against HPV 18 E6₁₂₁₋₁₃₅, classII-restricted, and therefore blockable with HB145) were co-cultured witha gp100 peptide pool, OKT3 antibody, or DCs pulsed with an HPV 18 E7peptide pool, HPV 18 E7 peptide pool and W6/32, HPV 16 E7₁₁₋₁₉, HPV 16E7₁₁₋₁₉ and W6/32, HPV 16 E7₁₁₋₁₉ and HB145, HPV 18 E6₁₂₁₋₁₃₅, HPV 18E6₁₂₁₋₁₃₅ and W6/32, or HPV 18 E6₁₂₁₋₁₃₅ and HB 145. The results areshown in FIG. 3. As shown in FIG. 3, TIL from tumor fragment F16 ofPatient 1 showed class I restricted recognition of HPV 18 E7 peptides.

Example 3

This example demonstrates the cloning of TIL from tumor fragment 16 ofPatient 1 to isolate HPV 18 E7 reactive CD8 positive T cells.

DCs were loaded with HPV 18 E7 and co-cultured with TIL from tumorfragment 16 (F16) of Patient 1. The TIL were sorted for 4-1BB positivecells using fluorescence activated cell sorting (FACS). The sorted cellswere cultured in 96-well plates with two cells per well. The clones werescreened for tumor reactivity against a gp100 peptide pool or a HPV 18E7 peptide pool. The results are shown in FIGS. 4A and 4B. As shown inFIGS. 4A and 4B, CD8 positive T cell cloning from tumor fragment F16using 4-1BB-based FACS sorting resulted in the isolation of two clones(12 and 21) with E7 peptide pool reactivity.

Example 4

This example demonstrates the reactivity of the TIL generated in Example1 from Patient 12.

TIL were generated as described in Example 1 from 36 different tumorfragments (F1-F36) from Patient 12. The TIL from Patient 12 or melanomaTIL (control) were co-cultured with dendritic cells pulsed with the HPV18 E6 peptide pool, the HPV 18 E7 peptide pool or a gp100 peptide pool(control) and IFN-γ was measured. The results are shown in FIG. 5. Asshown in FIG. 5, the TIL from the F1 and F15 tumor fragments fromPatient 12 showed the highest levels of IFN-γ production.

Autologous DCs were transduced with an HPV 18 E6 lentiviral vector or agreen fluorescent protein (GFP) lentiviral vector. Other autologouscells were pulsed with a gp100 peptide pool or a HPV 18 E6 peptide pool.Transduced cells were co-cultured with TIL from tumor fragment F1 ofPatient 12, or melanoma TIL 1, 2, or 3. The results are shown in FIG. 6.As shown in FIG. 6, TIL generated from tumor fragment F1 of Patient 12recognized DCs transduced with HPV 18 E6, suggesting that the TIL targeta naturally processed and presented antigen.

Example 5

This example demonstrates the reactivity of TIL clones from tumorfragments 1 and 15 of Patient 12 to isolate HPV 18 E6 reactive CD8positive T cells.

DCs were loaded with HPV 18 E6 and co-cultured with TIL from tumorfragments 1 and 15 of Patient 12. The TIL were sorted for 4-1BB positivecells using FACS. Cells were further sorted into CD4 positive and CD 8positive populations. The sorted cells were cultured in 96-well plateswith two cells per well. The clones were screened for tumor reactivityagainst a HPV 18 E6 peptide pool. Out of 480 wells of CD4 positive cellsfrom F1, 14 grew and 2 were reactive. Out of 912 wells of CD8 positivecells from F1, 33 grew and none were reactive. Out of 470 wells of CD4positive cells from F15, 163 grew and 32 were reactive. Out of 960 wellsof CD8 positive cells from F15, 41 grew and none were reactive.

The CD4 sorted cells were also tested for reactivity as measured bytumor necrosis factor (TNF) a secretion upon co-culture with a HPV 18 E6peptide pool (a pool spanning the entire E6 protein), no peptide,subpools of the HPV 18 E6 protein, or peptides 30-37 of the HPV 18 E6peptide pool. Each subpool contained a portion of the initial peptidepool. The results are shown in FIGS. 7A-7C. As shown in FIGS. 7A-7C,clones 3, 12, and 20 of tumor fragment F1 from Patient 12 were reactiveagainst HPV 18 E6. The CD4 positive T cell clones that were generatedrecognized two sequential 15-mers with an 11 amino acid overlap. Thepeptides shared the epitope HPV 18 E6125-135.

Example 6

This example demonstrates that the clones generated from the F15 tumorfragment of Patient 12 recognize HPV 18 E6₁₂₁₋₁₃₅ in an HLA-DRB1*15restricted manner.

Clones 3, 12, and 20 were co-cultured with donor PBMC with thehaplotypes set forth in Table B. The donor PBMC were pulsed with HPV 18E6₁₂₁₋₁₃₅.

TABLE B HLA-DR HLA-DQ HLA-DP Donor 1 13:02 15:01 06 06 04:02 03:01 Donor2 03:01 04:01 02 03:02 04:02 02:01 Donor 3 13:01 15 06 06 02:01 03:01Donor 4 03 07:01 02:01 02:02 04:01 01:01 Donor 5 01:02 14 05:01 05:0304:01 0501 Donor 6 01:01 13:02 05:01 06  5:02  9:01 Patient 12 03 15:0102 06:02 04:01 09

TNFα secretion was measured. The results are shown in FIGS. 8A-8C. Asshown in FIGS. 8A-8C, the clones generated from the F15 fragment ofPatient 12 recognized HPV 18 E6₁₂₁₋₁₃₅ pulsed onto PBMC that werematched at both HLA-DRB1*15 and HLA-DQB1*06, but not PBMC that werematched at only HLA-DQB1*06, suggesting HLA-DRB1*15 restriction. Thephenotypic allele frequency of HLA-DRB1*15 is 25 percent.

The TIL from clone 3 of tumor fragment F15 of Patient 12 wereco-cultured with autologous PBMC or donor PBMC pulsed with HPV 18E677-91 or HPV 18 E6₁₂₁₋₁₃₅. The results are shown in FIG. 9A. As shownin FIG. 9A, the TIL from clone 3 of tumor fragment F15 of Patient 12recognized PBMC matched only at DRB1*15 but not at only DRB1*06.

The TIL from clone 20 of tumor fragment F15 of Patient 12 wereco-cultured with HPV 18 E6₁₂₁₋₁₃₅ in the presence of antibodies againstHLA-DR, HLA-DQ, HLA-DP, pan-class I antibodies, or pan-class IIantibodies. Pan-class I and II antibodies block T cell binding to MHCClass I or Class II molecules, respectively. The results are shown inFIG. 9B. As shown in FIG. 9B, the recognition of cognate peptide by TILfrom clone 20 of tumor fragment F15 of Patient 12 was inhibited byblocking antibodies against HLA-DR. As shown in FIGS. 9A and 9B, the TILfrom tumor fragment F15 of Patient 12 recognize HPV 18 E6₁₂₁₋₁₃₅ in aDRB1*15 restricted manner.

Example 7

This example demonstrates the reactivity of TIL from Patients 4 and 8.

TIL were generated as described in Example 1 from 24 different tumorfragments (F1-F24) from Patient 4 or Patient 8. The TIL from Patient 4or melanoma TIL (control) were co-cultured with autologous DCs pulsedwith the HPV 16 E6 peptide pool, the HPV 16 E7 peptide pool or a gp100peptide pool (control) and IFN-γ was measured. The results are shown inFIG. 10. As shown in FIG. 10, the TIL from F4, F5, F14, F19, and F22tumor fragments were among those tumor fragments that showed reactivityagainst autologous DCs pulsed with HPV 16 E6 and E7 peptide pools.

The TIL from Patient 8 or melanoma TIL (control) were co-cultured withautologous DCs pulsed with the HPV 18 E6 peptide pool, the HPV 18 E7peptide pool or a gp100 peptide pool (control) and IFN-γ was measured.The results are shown in FIG. 13. As shown in FIG. 13, TIL weregenerated that showed reactivity against autologous DCs pulsed with theHPV 18 E7 peptide pool.

Example 8

This example demonstrates the cloning of TIL from tumor fragments ofPatient 4 to isolate HPV reactive CD4 and CD8 positive T cells.

DCs were loaded with HPV 16 E6 or HPV 16 E7 and co-cultured with TILfrom tumor fragments of Patient 4. The HPV 16 E6 and HPV 16 E7 reactiveTIL were separately sorted for 4-1BB positive cells using FACS. Thenumbers of cells were expanded as described in Example 1. Cells werefurther sorted into 4-1BB positive cells by FACS. The sorted cells werecultured in 96-well plates with two cells per well. Cells were furthersorted into CD4 positive and CD8 positive populations. The clones werescreened for tumor reactivity against a HPV 16 E6 or E7 peptide pool.

The results are shown in FIGS. 11A-11D. As shown in FIGS. 11A-11D, CD8positive and CD4 positive T cell clones with reactivity against HPV 16E6 and E7 were generated.

Example 9

This example demonstrates that adoptive cell therapy using anti-HPV Tcells treats cancer.

Inclusion Criteria for the study included (1) recurrent/refractory ormetastatic cervical cancer or high-risk HPV-positive cancer from anysite and (2) prior chemotherapy with platinum, including chemoradiation.

Tumors were resected from patients. TIL were obtained from the tumor,grown, the numbers of TIL were expanded, and the expanded numbers of TILwere screened for HPV reactivity as described in Example 1.

Patients received a non-myeloablative, lymphodepleting preparativeregimen of cyclophosphamide (60 mg/kg/day) intravenously (IV) on days −7and −6 and fludarabine (25 mg/m2/day) IV on days −5 through −1.

TIL were intravenously administered to the patients on Day 0. A highdose of aldeskeukin (interleukin (IL)-2) (720,000 IU/kg) wasintravenously administered to the patients on Days 0 through 4.

Patients underwent complete evaluation of tumor 4 to 6 weeks after thecompletion of the initial treatment regimen (defined as the last day ofaldesleukin administration). If the patient had stable disease or tumorshrinkage, repeat complete evaluations were performed monthly forapproximately 3-4 months, and then every 3-4 months until off studycriteria are met. All measurable lesions up to a maximum of 10 lesionsrepresentative of all involved organs were identified as target lesionsand recorded and measured at baseline. All other lesions (or sites ofdisease) were identified as non-target lesions and were also recorded atbaseline. Lesions were evaluated according to the Response EvaluationCriteria in Solid Tumors (RECIST) guideline (version 1.0) as set forthin Table C (target lesions) and Table D (non-target lesions).

TABLE C Complete Response (CR) Disappearance of all target lesionsPartial At least a 30% decrease in the sum of the longest diameterResponse (PR) (LD) of target lesions taking as reference the baselinesum LD. Progression At least a 20% increase in the sum of LD of targetlesions (PD) taking as reference the smallest sum LD recorded since thetreatment started or the appearance of one or more new lesions. StableNeither sufficient shrinkage to qualify for PR nor sufficient Disease(SD) increase to qualify for PD taking as references the smallest sumLD.

TABLE D Complete Disappearance of all non-target lesions Response (CR)and normalization of tumor marker level Non-Complete Persistence of oneor more non-target lesions Response Progression Appearance of one ormore new lesions. Unequivocal (PD) progression of existing non-targetlesions

Eleven patients were treated. The results are summarized in Table E.

TABLE E Response (duration Primary Prior systemic Cells IL-2 in PatientGender Age site Histology Disease sites therapy TIL site (x10⁹) dosesmonths)*  1 F 30 Cervix Adeno- Lungs, hilum, Cisplatin Lung 101 7 NRsquamous retroperitoneal, iliac, vaginal cuff  2 M 54 Tonsil SquamousNeck, soft tissue Docetaxel, cisplatin, Neck soft tissue 89 1 NR5-Fluorouracil (FU), cetuximab  3 F 53 Cervix Squamous Lungs, liver,pelvis Cisplatin, paclitaxel, Lung 126 3 PR (2) carboplatin, topotecan,ixabepilone, phase I  4 F 35 Cervix Squamous Mediastinum, hilum,Cisplatin, gemcitabine, Paratracheal 152 2 PR (9+) lung topotecan,paclitaxel node  5 F 55 Cervix Squamous Axilla, abdominal Carboplatin,5-FU, Axillary lymph 7 NR wall irinotecan node (LN)  6 M 60 UnknownSquamous Liver, neck, bone, Carboplatin, cisplatin, Liver 150 6 NR(neck) chest wall, capecitabine retroperitoneal, periportal  7 F 44Cervix Squamous Mediastinum, Cisplatin aortopulmonary 90 5 NRsupraclavicular, (AP) window brain LN  8 F 37 Cervix AdenoIntraperitoneal, Cisplatin right upper 75 8 PR (2+) retroperitoneal,quadrant (RUQ) abdominal wall. liver intraperitoneal surface  9 F 59Cervix Adeno Abdominal wall, lung Cisplatin, carboplatin, Lung 33 8Pending paclitaxel, bevacizumab 10 F 58 Unknown Squamous Mediastinum,hilum, Cisplatin, 5-FU, AP window LN 32 2 Pending (pelvis) lungcarboplatin, paclitaxel, cetuximab, irinotecan 11 F 31 Cervix Adeno-Perihepatic, pelvic Cisplatin, paclitaxel Pericecal squamousintraperitoneal NR = No response.

As shown in Table E, out of the eight patients for which results wereavailable, adoptive cell therapy with HPV reactive TIL resulted in threeobjective responders (OR), all of which were partial responders (PR).Two partial responses are ongoing at two months (Patients 3 and 4)following treatment and one partial response (Patient 8) is ongoing atnine months following treatment.

Computed tomography (CT) scans of the chest and pelvis of Patient 4 werecarried out before treatment and nine months after treatment. Theresults are shown in FIGS. 12A-F. As shown in FIGS. 12A-B, the cancerouslesion in the paraaortic lymph node had shrunk by 100% nine months aftertreatment. As shown in FIGS. 12C-D, the cancerous lesion in the leftlung hilar lymph node had also shrunk by 100% nine months aftertreatment. As shown in FIGS. 12E-F, the cancerous lesion in the commoniliac lymph node had also shrunk by 100% nine months after treatment.

Magnetic resonance imaging (MRI) scans of the liver of Patient 8 werecarried out before treatment and two months after treatment. The resultsare shown in FIGS. 14A-14B. As shown in FIGS. 14A-B, the cancerous masson the liver shrunk by 100% two months after treatment. CT scans of theabdomen and pelvis of Patient 8 were also carried out before treatmentand two months after treatment. The results are shown in FIGS. 14C-H. Asshown in FIGS. 14C-D, the cancerous lesion in the retroperitoneal lymphnode had also shrunk by 100%. As shown in FIGS. 14E-F, the cancerousmass in the abdominal wall had also shrunk by 100%. In addition, asshown in FIGS. 14G-H, the cancerous left pericolic mass shrunkdramatically.

Example 10

This example provides updated results of the clinical study described inExample 9 that were obtained nine months after the results described inExample 9 were obtained. This example demonstrates that adoptive celltherapy using anti-HPV T cells treats cancer.

Methods: A clinical trial to treat metastatic HPV+ cancers withtumor-infiltrating lymphocytes (TIL) selected for HPV E6- andE7-reactivity (HPV-TIL) was carried out as described in Example 9.HPV-TIL infusion was preceded by non-myeloablative conditioning andfollowed by high-dose bolus aldesleukin as described in Example 9.HPV-reactivity was assessed by ELISPOT, IFN-gamma production, and CD137expression assays.

Results: Nine cervical cancer patients were treated on the study. Theyreceived a median of 81×10⁹ T cells (range 33 to 152×10⁹) as a singleinfusion. The infused cells possessed reactivity against high-risk HPVE6 and/or E7 in 6/8 patients. The two patients with no HPV reactivitydid not respond to treatment. Three out of six patients with HPVreactivity demonstrated objective tumor responses by RECIST (1 PR and 2CR). One patient had a 39% best response. Two patients with widespreadmetastases had complete tumor responses that were ongoing 18 and 11months after treatment. One patient with a complete response had achemotherapy-refractory HPV-16+ squamous cell carcinoma (Patient 4 ofExample 9) and the other a chemoradiation-refractory HPV-18+adenocarcinoma (Patient 8 of Example 9). Both patients demonstratedprolonged repopulation with HPV-reactive T cells following treatment.Increased frequencies of HPV-specific T cells were detectable after 13months in one patient and 6 months in the other. Two patients withHPV-reactive TIL that did not respond to treatment did not displayrepopulation with HPV-reactive T cells.

Six non-cervical cancer patients were also treated on the study. Onepatient experienced an objective clinical response, that is, a partialresponse of a metastatic tonsil cancer that was ongoing four monthsafter treatment (FIGS. 20A-J).

The results are shown in Tables F-G.

TABLE F HPV-TIL cervical cancer cohort Response (duration HPV Cells IL-2in Patient Age Histology type Disease sites Prior systemic therapy TILsite (x10⁹) doses months)*  1 30 Adeno- HPV- Lungs, hilum, CisplatinLung 101 7 PD squamous 18 retroperitoneal, iliac, vaginal cuff  3 53Squamous HPV- Lungs, liver, pelvis Cisplatin, paclitaxel, Lung 126 3 PR(3) 18 carboplatin, topotecan, ixabepilone, phase I  4 35 Squamous HPV-Mediastinum, hilum, Cisplatin, gemcitabine, Paratracheal 152 2 CR (18+)16 lung, illiac topotecan, paclitaxel node  5 55 Squamous HPV- Axilla,abdominal wall Carboplatin, 5-FU, Axillary LN 81 7 PD 16 irinotecan  744 Squamous HPV- Mediastinum, Cisplatin AP window 90 5 PD 18supraclavicular, brain LN  8 37 Adeno HPV- Paracolic, retroperitoneal,Cisplatin RUQ 75 8 CR (11+) 18 abdominal wall, liver intraperitonealsurface  9 59 Adeno HPV- Abdominal wall, lung Cisplatin, carboplatin,Lung 33 8 PD 18 paclitaxel, bevacizumab 11 31 Adeno- HPV- Perihepatic,pelvic Cisplatin, paclitaxel Pericecal 46 9 PD squamous 18 12 37 AdenoHPV- Pelvis, retropertioneum, Carboplatin, paclitaxel, Supraclavicular70 1 PD 18 axilla, mediastinum, lung ipilimumab LN *Measured in monthsfrom cell infusion.

TABLE G HPV-TIL non-cervical cancer cohort Response Primary Cells IL-2(duration in Patient Gender Age site Histology Disease sites Priorsystemic therapy TIL site (x10⁹) doses months)* Head and neck  2 M 54Tonsil Squamous Neck, soft tissue Docetaxel, cisplatin, Neck 89 1 PD5-FU,cetuximab soft tissue  6 M 60 Unknown Squamous Liver, neck, bone,chest Carboplatin, cisplatin, Liver 150 6 PD (neck) wall,retroperitoneum, capecitabine periportal 13 M 60 Tonsil Squamous Lung,hilum Docetaxel, cisplatin, Lung 131 5 PR (4+) bevacizumab, cetuximab,gemcitabine Anal 10 F 58 Unknown Squamous Mediastinum, hilum, Cisplatin,5-FU, AP 32 2 PD (pelvis lung carboplatin, paclitaxel, window with AIN)cetuximab, irinotecan LN 14 F 49 Anal Squamous Mediastinum, 5-FU,mitomycin, Neck LN 69 3 PD retroperitoneum, pelvis cisplatin,carboplatin, protein-bound paclitaxel 15 F 58 Anal Squamous Liver,retroperitoneum, 5-FU, mitomycin, Liver 48 6 PD pelvis cisplatin *Measured in months from cell infusion.

These data show that HPV-TIL can mediate durable, complete regression ofmetastatic cervical cancer and that cellular therapy can mediatecomplete regression of an epithelial malignancy. These data also showthat HPV-TIL can mediate regression of a metastatic tonsil cancer.

Example 11

This example further describes the complete tumor responses obtained inExample 10 with adoptive cell therapy using anti-HPV T cells.

Methods

HPV-TIL generation: Tumor-infiltrating lymphocytes (TIL) were grown from2 mm fragments of excised tumors as described previously (Dudley et al.,J. Immunother., 26(4): 332-42 (2003)). After two to three weeks oflymphocyte outgrowth, the cultures were assessed for cellularcomposition by flow cytometry and for reactivity against HPVtype-specific E6 and E7 by interferon (IFN)-gamma production assay asdescribed in the Assessment of HPV oncoprotein reactivity section below.Flow cytometric analysis was performed with fluorescent antibodiesspecific for CD3, CD4, CD8, and CD56 (BD Biosciences). Cultures wereselected for additional expansion based on reactivity against the HPVoncoproteins, rapid growth, high T cell purity, and high frequency ofCD8+ T cells. Expansion to the cell numbers used for treatment wasaccomplished with a rapid expansion protocol with G-REX gas permeableflasks (Dudley et al., J. Immunother., 26(4): 332-42 (2003); Jin et al.,J. Immunother., 35(3): 283-92 (2012)). Infusion products were certifiedfor viable cell numbers, T cell purity (flow cytometry), potency (IFN-γproduction), sterility (microbiological studies), and absence of tumorcells (cytopathology).

Patient treatments: Patients had metastatic cervical cancer andmeasurable disease. Prior treatment with a platinum agent in either theprimary chemoradiation or metastatic setting was required. Theconditioning regimen consisted of cyclophosphamide 60 mg/kg IV daily fortwo days followed by fludarabine 25 mg/m² IV daily for five days. Cellswere administered IV over 20 to 30 minutes. Aldesleukin 720,000IU/kg/dose IV was initiated within 24 hours of cell infusion andcontinued every eight hours until stopped for toxicity or for a maximumof 15 doses. Filgrastim was initiated the day after cell infusion andcontinued until neutrophil counts recovered.

Tumor responses: Baseline imaging studies were obtained within fourweeks before initiating the conditioning regimen. Follow-up imaging wasobtained six weeks after treatment, monthly for three assessments, everythree months for three assessments, and then every 6 months for twoassessments.

Assessment of HPV oncoprotein reactivity: HPV reactivity was determinedby coculture of T cells (40,000 to 100,000 cells) with autologousimmature dendritic cells (40,000 cells) loaded with 1 μM of peptidepools spanning E6, E7, gp100, or EBNA1 and BZLF1 (Miltenyi Biotec,Bergisch Gladbach, Germany). Peptide pools included 15-mer peptidesoverlapping by 11 amino acids. Dendritic cells were generated from theadherent fraction of peripheral blood mononuclear cells (PBMC) or fromCD14+ cells isolated from PBMC using magnetic bead isolation (MiltenyiBiotec) by culturing in DMEM supplemented with 10% human serum and 1000IU/ml GM-CSF and 500 IU/ml IL-4 for five to six days. Anti-EBV control Tcells were generated before treatment by culturing PBMC with EBNA1 andBZLF1 peptide pools (10 μg/mL) in AIM-V/RPMI media supplemented with 10%human serum and 3000 IU/ml IL-2. For IFN-γ production assays, theconcentration of IFN-γ in the supernatants was determined afterovernight coculture (R&D Systems (Minneapolis, Minn.) or Thermo FisherScientific (Waltham, Mass.)).

ELISPOT (Mabtech (Cincinnati, Ohio)) analysis was performed according tothe manufacturer's instructions. Briefly, ELIIP plates (WAIPSWU fromMillipore (Billerica, Mass.)) precoated with capture antibody (clone1-D1K, Mabtech) were seeded with 10,000 effector cells and 40,000 targetcells. After 16 to 18 hours of incubation, IFN-γ secretion was detectedby addition of a biotinylated anti-IFN-γ antibody (7-B6-1 biotin,Mabtech) for two hours at room temperature. Following incubation withstreptavidin-alkaline phosphatase (Mabtech) for one hour, substratereagent (5-bromo-4-chloro-3′-indolyphosphate p-toluidine/nitro-bluetetrazolium chloride, Kirkegaard & Perry Laboratories, Inc.(Gaithersburg, Md.)) was added to allow spot formation. Spot formationwas stopped by rinsing with tap water. Spots were counted using anIMMUNOSPOT automated reader (Cellular Technology, Ltd. (Shaker Heights,Ohio)). ELIPSOT responses against E6 or E7 were defined as positive ifmore than two times the negative control and greater than 10 spots/well.

CD137 upregulation assays were performed by flow cytometric analysisafter 20 to 24 hour coculture (Wolff et al., Blood, 110(1): 201-10(2007)). Cells were labeled with fluorescent antibodies against CD137,CD4, CD8, and CD3 (BD Biosciences, San Jose, Calif.). They werecounterstained with propidium iodide (BD Pharmingen, Franklin Lakes,N.J.) prior to data acquisition with a BD FACSCANTO II cell analyzer (BDBiosciences). Data was analyzed with FLOWJO software, Mac version 10(TreeStar, Ashland, Oreg.).

Immunohistochemistry: Immunohistochemical stainings were performed inthe Laboratory of Pathology, NCI, on 4 μM sections from formalin-fixed,paraffin-embedded metastatic tumors according to standard procedures.After deparaffinization, rehydration, and antigen retrieval, tumorsections were incubated with anti-human CD4 clone 1F6 (Novocastra,Wetzlar, Germany) at a 1:80 dilution for 2 hours, anti-human CD8 cloneCD8/144B (Dako Corp., Glostrup, Denmark) at a 1:50 dilution for 2 hours,or anti-human p16 clone JC8 (Santa Cruz, Dallas, Tex.) at a 1:200dilution for 32 minutes. The CD4 stained slides were stained on anAUTOSTAINER Link 48 (Dako Corp.) and visualized with the ENVISION FLEX+detection system (Dako Corp.). The CD8 and p16 stained slides werestained on a VENTANA Benchmark XT (Ventana Medical Systems, Tucson,Ariz.) and visualized with the ULTRAVIEW detection system (VentanaMedical Systems). Images were captured with 10× microscopy.

Determination of lymphocyte subsets from peripheral blood: Completeblood counts with manual differential determination were performed bythe Clinical Center Hematology Laboratory. Lymphocyte phenotyping for T,B, and NK cells was performed by the NIH Immunology Flow CytometryLaboratory using standardized criteria.

Real-time reverse transcription polymerase chain reaction (RT-PCR): RNAwas isolated from a 2 mm fragment of fresh tumor tissue using an RNEASYkit (Qiagen, Valencia, Calif.). Reverse transcription first-strand DNAsynthesis was performed using QSCRIPT cDNA supermix (Quanta BioSciences,Gaithersburg, Md.). Custom made TAQMAN primer and probe sequences(Applied Biosciences, Foster City, Calif.) were used for HPV16-E6,HPV16-E7, HPV18-E6, and HPV18-E7. Readily availableglyceraldehyde-3-phosphate dehydrogenase (GAPDH) primer probe set wasused to standardize oncoprotein expression levels (Hs02758991_g1,Applied Biosciences, Foster City, Calif.). RT-PCR was performed on a7500 FAST REAL-TIME PCR System (Applied Biosciences).

Analysis of serum cytokine levels: Levels of 17 cytokines (IL-1β, IL-2,IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17,granulocyte-colony stimulating factor (G-CSF), granulocytemonocyte-colony stimulating factor (GM-CSF), IFN-γ, monocyte chemotacticprotein (MCP)-1, macrophage inflammatory protein (MIP)-1β, and tumornecrosis factor (TNF)-α were measured in sera from patients collectedbefore and after treatment with HPV-TIL using BIO-PLEX Pro HumanCytokine 17-plex Assay (Bio-Rad Laboratories) according to themanufacturer's instructions. The cytokine levels were acquired by theBIO-PLEX 200 system (Bio-Rad).

Case Reports

Patient 4 was diagnosed with stage 3B poorly-differentiated, squamouscell cervical cancer fourteen months before treatment with HPV-TIL. Thepatient was initially treated with cisplatin, vincristine, and bleomycinfollowed by chemoradiotherapy with gemcitabine plus cisplatin, andbrachytherapy. Two months later, metastatic cancer was detected inparatracheal (biopsy-confirmed), subcarinal, and bilateral hilar lymphnodes. She received four cycles of topotecan and paclitaxel beforedisease progression, and then was referred for the clinical trialdescribed in Examples 9 and 10. HPV-TIL was prepared from a resectedparatracheal lymph node. The patient received lymphocyte-depletingchemotherapy followed by a single intravenous infusion of 152×10⁹HPV-TIL and two doses of aldesleukin. Aldesleukin dosing was stopped forpatient fatigue. She was discharged from the hospital afterhematological recovery, 12 days after cell infusion.

Patient 8 was diagnosed with stage IB2 adenocarcinoma of the uterinecervix 17 months before treatment with HPV-TIL. Her primary tumor wastreated with chemoradiation with cisplatin followed by brachytherapy.Five months later, she was noted to have a chemoradiation-refractoryprimary tumor (biopsy-confirmed). Salvage surgery identified paraaorticand iliac lymph node involvement and residual pelvic disease. Her cancerprogressed to involve additional retroperitoneal lymph nodes and theliver surface, and she developed right hydroureteronephrosis andbilateral pulmonary emboli, which required a ureteral stent andanticoagulation therapy. The patient was then treated according to theprotocol described in this Example using HPV-TIL generated from twoperitoneal nodules. She received lymphocyte-depleting chemotherapyfollowed by 75×10⁹ HPV-TIL cells and eight doses of aldesleukin.Aldesleukin dosing was stopped for hypoxia secondary to pulmonary edema,which required supplemental oxygen and resolved with diuresis. Dischargefrom the hospital was 11 days after cell infusion.

Complete Clinical Responses

Both patients had disseminated progressive disease before treatment(FIG. 12A-F; FIG. 14A-H; FIG. 15 A-B; FIG. 16A-D; FIG. 21A-H; and FIG.22A-H). Patient 4 had metastatic tumors involving a paraaorticmediastinal lymph node, bilateral lung hila, subcarinal lymph nodes, andiliac lymph nodes (FIG. 15 A; FIG. 12A-F; and FIG. 21A-H). Patient 8 hadmetastatic cancer involving at least seven sites: two tumors on theliver surface, paraaortic and aortocaval lymph nodes, the abdominalwall, a pericolic mass in the left pelvis, and a nodule obstructing theright ureter (FIG. 15 B; FIG. 14A-H; FIG. 16A-D; and FIG. 22A-H). Eachpatient was treated with a single infusion of T cells, which resulted intumor regression that occurred over months (FIG. 15A-B). Both patientsexperienced objective complete tumor responses, which were ongoing 18and 11 months after treatment (FIG. 21A-H (Patient 4) and FIG. 22A-H(Patient 8). A previously placed ureteral stent was removed from Patient8 following regression of the tumor obstructing her right ureter (FIGS.22 G and H). Neither patient received additional therapy. Both patientshave returned to full-time employment.

Toxicity of HPV-TIL

There were no acute toxicities related to cell infusion. No autoimmuneadverse events occurred. Both patients displayed transient serumcytokine elevations (FIG. 17A-B) that were associated with fevers, butneither patient developed severe cytokine release syndrome. The levelsof cytokines in cryopreserved serum were determined. Testing was for thefollowing cytokines: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12(p70), IL-13, IL-17, G-CSF, Granulocyte macrophagecolony-stimulating factor (GM-CSF), IFN-γ, MCP-1, Macrophageinflammatory protein 1 beta (MIP-1β), and TNF-α. Cytokines with levelsgreater than twofold baseline on two consecutive measurements aredisplayed. Aldesleukin was dosed every eight hours after cell infusion(Patient 4 received two doses and Patient 8 received eight doses). GCSFwas administered daily beginning the day after cell infusion andcontinued until neutrophil counts recovered (Patient 4 received 11 dosesand Patient 8 received nine doses).

Aldesleukin was dosed to tolerance by protocol design, stopping forfatigue in Patient 4 and dyspnea in Patient 8. Grade 3 and grade 4adverse events are listed in Table H. The most common toxicities werehematological and the expected effects of the lymphocyte-depletingconditioning regimen (cyclophosphamide and fludarabine).

TABLE H Toxicity Patient 4 Patient 8 Anemia X X Neutropenia X XLymphopenia X X Leukopenia X X Thrombocytopenia X X Febrile NeutropeniaX X Infection X Fatigue X Nausea/Vomiting X X Syncope^(†) X Lowergastrointestinal (GI) X hemorrhage* Hematuria* X Hypophosphatemia X^(†)A single episode of unknown etiology 15 days after treatment.*Associated with radiation cystitis and colitis.

Tumor Antigen Expression and T Cell Infiltration

Metastatic tumors excised for the generation of HPV-TIL were a squamouscell carcinoma from Patient 4 and an adenocarcinoma from Patient 8. Themalignant cells expressed p16INK4A, a sensitive indicator of high-riskHPV-infection. The HPV type and the expression levels of E6 and E7, thetarget antigens of HPV-TIL, were determined for each patient's tumor byreal-time reverse transcription polymerase chain reaction (RT-PCR).Patient 4 had a HPV-16+ cancer and Patient 8 had a HPV-18+ cancer. The Tcell infiltrate in tumors from both patients showed a mixed compositionwith predominantly CD8+ cells in Patient 4 and CD4+ cells in Patient 8.Both CD4+ and CD8+ T cells grew from the excised tumors. The infusedHPV-TIL were composed of 19% CD4+ and 79% CD8+ T cells for Patient 4,and 15% CD4+ and 87% CD8+ T cells for Patient 8.

HPV Oncoprotein Targeting by HPV-TIL

The HPV-TIL administered to Patient 4 were reactive against both the E6and E7 oncoproteins as demonstrated by interferon (IFN)-γ production andELISPOT assays (FIGS. 18A and C). Five percent and greater than sevenpercent of the infused cells showed responses to E6 or E7, respectivelyby ELISPOT assay (FIGS. 18C and D). E6 responses were CD8+ Tcell-mediated, and E7 responses were CD4+ and CD8+ T cell-mediated. Intotal, 14 percent of the infused cells displayed HPV reactivity asmeasured by CD137 upregulation assay. For Patient 8, HPV-TIL werereactive against E7 (FIG. 18B), with four percent of T cells respondingto the antigen by ELISPOT assay (FIG. 18D). This response was primarilymediated by CD4+ T cells.

Repopulation with Oncoprotein Reactive T Cells

HPV-TIL infusion was followed by rapid increases in peripheral bloodCD4+ and CD8+ T cells but not NK and B cells (FIG. 19A-B). Expansion ofthe numbers of infused T cells was associated with establishment andpersistence of peripheral blood T cell reactivity against the HPVoncoproteins as measured by IFN-γ production, ELISPOT, and CD137upregulation assays (FIG. 19C-F). Both patients had little, if any,reactivity against E6 or E7 prior to treatment. Following treatment,Patient 4 acquired robust T cell recognition of E6 and E7. For Patient8, this recognition was weaker but nonetheless detectable and,consistent with the infused T cells, directed against only E7. One-monthafter treatment, 12 percent of Patient 4's peripheral blood T cells wereoncoprotein reactive (seven percent against E6 and five percent againstE7) (FIG. 19E). Reactivity against these antigens was sustained with onepercent of peripheral blood T cells showing oncoprotein recognition fourand 13 months after cell infusion (FIG. 19E). Patient 8 showed 0.4percent HPV reactive T cells one-month after treatment (FIG. 19F). Thisreactivity was sustained, albeit at lower levels, three and six monthsafter treatment (FIGS. 19 D and F). Consistent with the reactivity ofthe T cell subsets in the infused HPV-TIL, the HPV specific T cells thatrepopulated the patients were primarily E6 and E7 reactive CD8+ T cellsfor Patient 4, and E7 reactive CD4+ T cells for Patient 8.

Example 12

This example provides updated results of Patients 4 and 8 from theclinical study described in Examples 10 and 11 that were obtained fourmonths after the results described in Examples 10 and 11 were obtained.This example demonstrates that adoptive cell therapy using anti-HPV Tcells treats cancer.

The objective complete tumor responses of Patients 4 and 8, who weretreated as described in Examples 10 and 11, were ongoing 22 and 15months after treatment, respectively.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A method of treating or preventing cervicalcancer in a patient, the method comprising: (a) culturing multiplefragments of an HPV⁺tumor sample from the patient in the presence ofinterleukin-2 (IL-2) (b) obtaining T-cells from the cultured fragments;(c) expanding the number of T-cells to produce an expanded population ofHPV-specific T-cells using one or both of (i) irradiated allogenicfeeder cells and (ii) irradiated autologous feeder cells; and one orboth of (iii) OKT3 antibody and (iv) IL-2, wherein the T-cells have notbeen depleted of CD4⁺cells; (d) optionally, adding a second culturingstep following (c); and (e) administering the expanded population ofHPV-specific T-cells to the patient in an amount effective to treat orprevent the cervical cancer in the patient.
 2. The method of claim 1,wherein a second culturing step is added after step (c) to furtherexpand the number of HPV-specific T-cells.
 3. The method of claim 1,wherein the IL-2 in step (a) is present at 6000 IU.
 4. The method ofclaim 1, wherein the IL-2 in step (c) is present at 6000 IU.
 5. Themethod of claim 1, further comprising, prior to step (a), obtaining anHPV⁺tumor sample from the patient and fragmenting said tumor samples. 6.The method of claim 5, wherein the tumor samples are fragmentedmechanically or enzymatically.
 7. The method of claim 1, wherein theexpanded population of HPV-specific T-cells recognizes HPV 16-positivecervical cancer cells.
 8. The method of claim 1, wherein the expandedpopulation of HPV-specific cells recognizes an HPV antigen selected fromthe group consisting of HPV 16 E6 and HPV 16 E7.
 9. The method of claim1, wherein the expanded population of HPV-specific cells recognizes anHPV antigen selected from the group consisting of HPV 18 E6 and HPV 18E7.
 10. The method of claim 1, further comprising administering to thepatient nonmyleoablative lymphodepleting chemotherapy prior to step (e).11. The method of claim 1, wherein the expanded population of cellsobtained in step (c) comprises multiple T-cells having different HPVspecificities.
 12. The method of claim 1, wherein the expandedpopulation of HPV-specific T-cells obtained in step (c) secretes atleast about 200 pg/mL of interferon-gamma.
 13. The method of claim 1,wherein the expansion step occurs over about 10 to about 14 days. 14.The method of claim 12, wherein the expanded population of HPV-specificT-cells is increased at least about 1000-fold to about 3000-fold. 15.The method of claim 1, wherein the number of HPV-specific T-cellsadministered to the patient is about 1×10¹⁰ to about 13.7×10^(10.) 16.The method of claim 1, wherein the expanded population of HPV-specificT-cells is administered with a pharmaceutically acceptable carrier. 17.The method of claim 16, wherein the pharmaceutically acceptable carrieris saline, 5% dextrose in water, Ringer's lactate, an electrolytesolution, or PLASMA-LYTE A.
 18. The method of claim 1, wherein theexpanded population of HPV-specific T-cells is enriched for CD4⁺T-cells.19. The method of claim 1, wherein the culturing step (a) is performedfor at least about 12 days.